The long-term goal of this project is to continue to investigate the dynamics of phospholipid-protein interactions and relate them to specific membrane functions. We propose to study, in detail, the role of lipid fluidity and phase separation on the activity of two regulatory membrane enzymes, namely the (Na plus K)-ATPase and the adenylcyclase. Our working hypothesis it that membrane fluidity, as defined by lipids, divalent metals, interacting proteins, and the phenomena of phase separation and clustering into separate domains is an important parameter in cell regulatory mechanisms. In parallel to these functional studies, we propose to investigate with several complementary techniques, structural aspects of the interaction of four purified and characterized membrane proteins with purified phospholipids, and investigate in detail the role of divalent metal ions and proteins on membrane fusion phenomena. Our approach is a synthetic one, based on the re-constitution of purified components, enabling us to study at the molecular level, structure-function relationships not otherwise amenable to detailed investigation. Our experimental system combines both biochemical techniques, such as purification, organic synthesis, enzymatic characterization, and also biophysical techniques such as differential scanning calorimetry, ion transport, isopiezic monolayer expansion, freeze-fracture electron microscopy, polarization of fluorescence, and x-ray diffraction. BIBLIOGRAPHIC REFERENCES: Jacobson, K. and Papahadjopoulos, D. Effect of a phase transition on the binding of ANS to phospholipid membranes. Biophys. J. 15, 549-565, 1976. Poste, G. and Papahadjopoulos, D. Lipid vesicles as carriers for introducing materials into cultured cells: Influence of vesicle lipid composition on mechanism of vesicle incorporation into cells. Proc. Nat. Acad. Sci. (USA) 73, 1603-1607, 1976.